Programmed cell death has been shown to be an essential feature of negative selection of autoreactive lymphocytes and regulation of both physiological and pathological immune responses. Fas, also known as CD95, is a member of the TNF-receptor superfamily and has been shown to be important in apoptosis of activated T and B lymphocytes initiated by signaling through their antigen receptors. Humans and mice with germ line dominant-negative mutations in Fas accumulate abnormal lymphocytes and develop systemic autoimmunity similar to patients with Systemic Lupus Erythematosus (SLE). While most patients with non-familial autoimmune disease do not carry Fas mutations, there is evidence that Fas-mediated apoptosis may be impaired in the milieu of chronic inflammation. We are investigating which signals regulate Fas-mediated apoptosis in T cells, with the eventual aim of harnessing these discoveries to modulate Fas-induced apoptosis for therapeutic goals in human disease. In activated CD4+ T cells, TCR restimulation triggers apoptosis that depends in large part on interactions between the death receptor Fas and its ligand FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but it is not known which pathways mediate this process. Using a variety of approaches, we are investigating molecular and cellular mechanisms regulating the TCR- and Fas-induced apoptosis pathways. We have found considerable heterogeneity in the ability of various T cell subsets to undergo Fas-mediated apoptosis and are investigating the molecular mechanisms underlying this heterogeneity. The goal in understanding these mechanisms is to design specific therapies to sensitize autoreactive lymphocytes to Fas-mediated apoptosis, which could constitute a long-acting and potentially permanent treatment for various autoimmune diseases such as SLE, Multiple Sclerosis, Rheumatoid Arthritis, Type-I diabetes, and others in which autoreactive lymphocytes play a role. Through collaborations with investigators at NIH studying patients with the Autoimmune Lymphoproliferative Syndrome (ALPS), a rare disorder associated with dominant-interfering Fas mutations, and the more common polygenic autoimmune disease SLE, we are investigating translational implications of these findings. We are also investigating the subcellular trafficking of Fas Ligand (FasL), the TNF-family cytokine ligand for Fas. In addition to trafficking to the plasma membrane as a type II transmembrane protein, FasL is known to be sorted into secretory lysosomes, where it can be secreted in vesicles and cleaved by metalloproteases. We are investigating which forms of FasL participate in RICD, and which molecules and motifs within the FasL cytoplasmic N-terminal domain direct its trafficking to secretory lysosomes. In collaboration with Ken Smiths laboratory at the University of Cambridge, we used systems biology to study the impact of polymorphisms in genes encoding TNF-family cytokines and their receptors in aggregate on susceptibility to autoimmune and inflammatory disease.